1. Field of the Invention
This invention relates to a process for manufacturing high-strength, spherical hydrated gel containing microorganism immobilized therein.
2. Description of the Prior Art
Studies have been under way on immobilization of enzymes or microorganisms to efficiently utilize their functions as biocatalysts, and some of the results have been put to practical use.
As a method of immobilizing enzymes or microorganisms is known the entrapped immobilization technique, in which enzymes or microorganisms are retained inside a polymeric material. The polymers frequently used for this purpose include agar, salts of alginic acid, K-carrageenan, polyacrylamide, polyvinyl alcohol and photo-curable polymers. Of these, polyvinyl alcohol (hereinafter abbreviated as PVA in some cases) can be converted into gel of high water content having excellent water resistance, elasticity and flexibility if its aqueous solution is frozen at a temperature not higher than -5.degree. C., followed by thawing at ordinary temperature, and this freeze-thaw gel can be used as a desirable carrier for immobilizing enzymes and microorganisms [Japanese Patent Kokoku No. 47-12854 (1972)]. It is also known that this PVA gel shows sufficiently high mechanical strength not to be expected with other polymers, if the freeze-thaw operation is repeated or if the freezing operation is followed by vacuum dehydration [Japanese Patent Kokai No. 58-47492 (1983)].
The polymeric materials used for immobilizing microorganisms must be nontoxic and have no adverse effect upon microorganisms. In this respect, freeze-thaw PVA gel shows high safety to living bodies because no chemical agent is used for gel formation. In addition, its high water content and porous structure make it ideal for use as a carrier suitable for the culture and propagation of the microorganisms immobilized therein.
Reactors in which bioreactions are carried out in the presence of an enzyme or microorganism immobilized by this entrapped method are various in type: fixed bed (packed column), fluidized bed, and stirrer-equipped tank. The shape of carrier used in these reactors should preferably be spherical in terms of large active area, high fluidity and packing efficiency, and ease of handling.
For commercial production, it is important that high-strength, spherical carriers be manufactured by simple operations and at a low cost.
Salts of alginic acid, which have also been used extensively as a low-cost and easy-to-handle carrier for immobilizing enzymes or microorganisms, readily form spherical gel beads when an aqueous solution of sodium alginate is added dropwise at ordinary temperature to an aqueous solution containing polyvalent metal ions, such as Ca.sup.2+ and Al.sup.3+. However, gel beads of this type are damaged by a salt of phosphoric acid, and tend to be eroded or dissolved when used for waste water treatment. In addition, these are liable to destruction when used over long periods in a reactor because of the insufficient mechanical strength.
Gels formed from photo-curable resins and polyacrylamide can also be shaped into spheres at ordinary temperature, but their practical application as carrier for entrapped immobilization is limited only to bioreactions which yield products of high added value [Japanese Patent Kokai No. 61-216688 (1986)] because of the far higher material cost compared with PVA gel.
PVA gel by freeze-thaw method may preferably be molded into spheres when used as carrier for immobilizing microorganisms. However, no suitable method has yet been established to mass-produce by simple operations spherical PVA gel beads with a diameter in the range from 1 to 10 mm--a shape most preferred for the purpose.
Various methods are known for molding PVA gel into spheres: putting an aqueous solution of PVA in a spherical mold and subjecting to freeze-thaw treatment [Japanese Patent Kokai No. 58-47492 (1983)]; adding an aqueous solution of PVA dropwise to a recessed, hemi-spherical mold immersed in a liquid, such as oil and organic solvents, and subjecting the whole system to freeze-thaw treatment [Japanese Patent Kokoku No. 54-1501 (1979)]; adding an aqueous solution of PVA to a dispersant, such as oil and organic solvents, with stirring, thereby forming microspheres of PVA gel with a diameter in the range from 0.1 .mu.m to 1 mm [Japanese Patent Kokai No. 62-45637 (1987)]; and adding an aqueous solution of PVA containing enzymes or microorganisms dropwise to saturated solution of boric acid to form spheres of PVA gel containing enzymes or microorganisms [Japanese Patent Kokai No. 61-100193 (1986)], and furthermore followed by freeze-thaw treatment [Japanese Patent Kokai No. 61-139385 (1986)].
In addition, Japanese Patent Kokai No. 62-138193 (1987) discloses "a process for manufacturing granular moldings containing enzymes or microbial cells immobilized therein, which comprises dropwise addition of an aqueous composition containing (A) polyvinyl alcohol, (B) a water-soluble polysaccharide capable of gelation upon contact with at least one member of polyvalent metal ions and (C) an enzyme or microbial cells to an aqueous medium containing polyvalent metal ions and boric acid, thereby gelling said aqueous composition into granules."
The above-mentioned methods using molds require the same number of molds as that of spherical moldings to be manufactured and freezing devices, and hence are not desirable for continuous production of spherical PVA gel in large quantities in terms of installation and freezing costs.
On the other hand, the methods using organic solvents, oil and chemical agent such as boric acid, are also undesirable and have limited use because of possible adverse effects of these chemicals upon microorganisms and possible contamination of PVA gel with impurities.